1. Field of the Invention
The present invention relates to a chimeric protein expressed from a transgene tranduced into tumor cells. Particularly, the invention pertains to a chimeric protein which, when expressed on tumor cells, can promote T cell activation without relying on the concurrent presence of either tumor-associated antigens or MHC molecules. Such T cell activation can lead to lysis of the tumor cells as well as bystander killing of non-transduced tumor cells in the vicinity and are therefore of great significance in cancer treatment.
2. Description of the Related Art
It is known in the art that cytotoxic T cells can recognize and kill tumor cells that express peptides derived from tumor associated antigens on their surface in association with MHC class I molecules. The identification of a wide range of tumor associated antigens from melanoma and other tumor cells has generated interest in developing strategies that employ activated CD8+ T cells for selective killing of tumor cells. Many tumors, however, display defects; in antigen processing and presentation. Stable expression of MHC class I molecules on the cell surface requires proteolytic generation of peptides by the proteosome in the cytosol and subsequent delivery of cytosolic peptides to the endoplasmic reticulum by the peptide transporters TAP1 and TAP2 (Goldberg and Rock 1992). Loss or downregulation of proteosome subunits, TAP-1, TAP-2, xcex22-microglobulin or MHC class I heavy chain have been documented for a wide range of solid tumors including melanoma (Maeurer et al. 1996) and prostate (Blades et al. 1995), lung (Korkolopoulou et al. 1996), bladder (Nouri et al. 1994), renal (Luboldt et al. 1996), colorectal (Kaklamanis et al. 1994), and breast (Vitale et al. 1998) carcinomas. Defective presentation of peptides by MHC class I molecules can allow tumor escape from immune recognition by CD8+ T cells (Restifo et al. 1996).
It is also known in the art that to overcome defects in antigen processing and presentation in tumor cells, cytotoxic T cells can be targeted to lyse tumor cells in a way that does not rely on the presence of MHC molecules on the tumor cells. One such method is through the use of chimeric antigen receptors expressed on T-cells. Such chimeric receptors have a single-chain antibody (scFv) fused to the zeta chain of the TCR CD3 complex and can be expressed on T-cells (Moritz et al. 1994; Altenschmidt et al. 1997; Altenschmidt et al. 1997; Alvarez Vallina et al. 1997). Another method is by intravenous administration of bispecific antibodies. Bispecific antibodies, that is, antibodies with specificity for both the CD3 molecule of T cells and surface antigens of tumor cells, can redirect activated T cells to attack and lyse tumor cells (Bolhuis et al. 1991). However, application of either method relies on the identification of tumor associated antigens that are preferentially expressed on the surface of tumor cells. Moreover, intravenous administration of bispecific antibodies can induce systemic cytokine release and toxicity (Weiner et al. 1995; Tibben et al. 1996).
The present invention discloses an alternative approach to promote T cell activation and lysis of tumor cells that is independent of the expression of antigens or MHC molecules on the tumor cells. This method, unlike the prior art methods, does not rely on the identification of any tumor associated antigens and does not induce systemic cytokine release and toxicity.
In accordance of the present invention, activation of T cells as a means of destroying tumor cells can be achieved by one or more chimeric surface proteins expressed from transgenes transduced into in vivo tumor cells or into ex vivo tumor cells which are then injected back in the tumor. Expression of the chimeric proteins on tumor cells can led to T cell activation, further resulting in lysis of the tumor cells. Thus, the chimeric proteins can be employed to destroy tumor cells in vivo, forming the basis of a new method for treating cancers.
As a preferred embodiment of the present invention, a chimeric protein, 2C11-xcex31-B7, has shown a significant T-cell activating property when expressed on mammalian cells in vivo. Further, when co-expressed with costimulatory molecules such as CD80 or CD86, the potency of 2C11-xcex31-B7 activation of T cells increases by several hundred folds. Also of significance is the inclusion of a spacer xcex31 (the hinge-CH2xe2x80x94CH3 region of human IgG1) between the effector (here, 2C11 single chain antibody) and anchor (B7, composed of the transmembrane domain and cytoplasmic tail of murine B7-1). The effector has the ability to activate T cells while the anchor is responsible for attaching the effector to the cell membrane. The spacer, on the other hand, can prevent or reduce proteolytic cleavage of the effector from the anchor. The method of activating T cells of the present invention is not limited to the particular chimeric proteins employed in the preferred embodiment, other proteins may be satisfactorily used as long as they can be expressed on the tumor cell surface.
Another objective of the invention is to more efficiently express chimeric proteins on the surface of mammalian cells in vivo. One major limitation on high level expression of chimeric surface proteins is proteolytic cleavage, which may in some cases reduce the expression to an undetectable level. This objective is achieved by introducing a spacer between the effector (i.e., the functional domain for T cell activation) and the anchoring transmembrane (TM) domain. Such arrangement dramatically reduces the proteolytic cleavage that usually occurs between the functional domain, such as scFv (2C11) and the TM. Of course, the invention is not limited to xcex31, which is used as an example in the preferred embodiment, other spacers may be satisfactorily used.
Still another objective of the invention is developing a method for treating cancers in mammals. This objective is achieved by using one of known gene transduction methods, such as direct injection of the transgenes into the tumors under treatment or through a viral delivery method (adenovirus or retrovius), to introduce transgenes into the tumor cells whereby the chimeric surface proteins with capability of activating T cells can be expressed. The specific transgene delivery method is not a limitation to the present invention. Any efficient existing or future developed technologies may be employed. For example, the following references disclose several transgene delivery methods employed in various situations:
Nagamachi Y, et al., xe2x80x9cSuicidal gene therapy for pleural metastasis of lung cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase genexe2x80x9d, Cancer Gene Ther. 6:546-53 (1999), where a transgene-encoding plasmid was mixed with liposomes to form a DNA-liposome complex. The mixture was injected interpleurally to mice that had lung cancer cells growing in the pleural cavity. The transgene was expressed in about 14% of the tumor cells in the pleural cavity.
Cao G, et al., xe2x80x9cAnalysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapyxe2x80x9d, Cancer Gene Ther. 6:572-80 (1999), where a retrovirus vector was constructed such that the gene of interest is expressed under the control of the CEA promoter. Nude mice that had established i.p. human colorectal carcinoma tumors were i.p. injected with retrovirus-producing cells, and the transgene was expressed in the tumors.
Puhlmann M, et al., xe2x80x9cVaccinia as a vector for tumor-directed gene therapy: biodistribution of a thymidine kinase-deleted mutantxe2x80x9d, Cancer Gene Ther. 7:66-73 (2000), where an attunuated replication-competent vaccinia virus was used as a vector for in vivo tumor transduction. The vaccinia virus was administered interperitoneally to mice that had established i.p. tumors. The reporter gene expression in i.p. tumors was up to 7000 times higher than in normal tissues. Intraveneous administration of the vaccinia virus resulted in much higher transgene expression (up to 180,000 fold difference) in sub cutaneous tumors compared to normal tissues.
Gambotto A, et al. xe2x80x9cInduction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12xe2x80x9d, Cancer Gene Ther. 6:45-53 (1999), where an adenoviral vector was constructed to express a gene under the control of the early cytomegalovirus immediate-early promoter, and the recombinant adenovirus were directly injected into murine tumors.
Gunji Y, et al. xe2x80x9cInhibition of peritoneal dissemination of murine colon carcinoma cells by administrating retrovirus harboring IL-2 genexe2x80x9d, Cancer Gene Ther. 5:339-43 (1998), where a retrovirus was injected into mouse i.p. tumors in vivo in which it expressed the gene of interest and showed therapeutic benefits.
Shirakawa T, et al., xe2x80x9cIn vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapyxe2x80x9d, Cancer Gene Ther. 5:274-80 (1998), where a recombinat adenoviral vector was constructed such that the gene of interest is expressed under the control of an osteocalcin promoter, and was then i.v. injected into nude mice that had established osteosarcoma metastasis in their lungs, resulting a specific expression of a reporter gene in the lung tumors of such mice.
Kwong Y L, et al., xe2x80x9cAdenoviral-mediated suicide gene therapy for hepatic metastases of breast cancerxe2x80x9d, Cancer Gene Ther. 3:339-44 (1996), where breast cancer tumors were established in the livers of mice. The gene of interest was cloned into a replication defective adenoviral vector. The vector was directly injected into tumors where it allowed expression of the transgene.
Ichikawa T, et al., xe2x80x9cIn vivo efficacy and toxicity of 5-fluorocytosine/cytosine deaminase gene therapy for malignant gliomas mediated by adenovirus. Cancer Gene Ther. 7:74-82 (2000), where a transgene of interest was placed in a vector and used to make adenovirus that carried the gene. The resulting adenovirus was directly injected into a brain tumor in rats where the tumor cells were infected with the virus, thereby expressing the gene.
Of course, there are possibly other suitable transgene delivery methods, either existing now or to be developed in the future. The specific transgene delivery method used, however, forms no part of the invention of cancer treatment.
Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the description and drawings are provided solely for purposes of illustration and not as a definition of the limits of the invention, for which reference should be made to the claims.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.